Currently, many high-value chemicals or fuels are typically manufactured by thermochemical processes from hydrocarbons, including petroleum oil and natural gas. Also, high value chemicals may be produced as “by-products” during the processing of crude oil into usable fractions. For example, isoprene has typically been produced during the catalytic cracking of crude oil fractions. However, recently catalytic cracker users have shifted their focus from crude oil to natural gas, resulting in a reduced source of the four and five carbon chain molecules that are found in crude oil, but not natural gas.
Being a short-chain carbon molecule, isoprene is a useful starting material for synthesizing a variety of chemicals. Isoprene may be used as a monomer or co-monomer for the production of higher value polymers. Examples of chemicals that can be produced using isoprene include polyisoprene, polybutylene, styrene-isoprene-styrene block co-polymers, and others. An example of an industry that uses isoprene is the synthetic rubber industry.
Given the increasing demand, decreasing supply and the many uses of isoprene, a new method of isoprene production is desired. Also, as the concerns of energy security, increasing oil and natural gas prices, and global warming escalate, the chemical production industry is seeking ways to replace chemicals made from non-renewable feedstocks with chemicals produced from renewable feedstocks using environmentally friendly practices.
The biological production of isoprene has been studied since the 1950s (Sharkey, T. D. 2009. The Future of Isoprene Research. Bull. Georg. Natl. Acad. Sci. 3: 106-113). Although many different organisms are known to emit isoprene, so far the biochemical pathway for isoprene production has only been elucidated in a few plant species. In plants, it appears that isoprene is produced in the chloroplast or other plastids from dimethylallyl diphosphate, also referred to herein as dimethylallyl pyrophosphate (DMAPP), in a single step by isoprene synthase, a nuclearly encoded enzyme that is routed to the plastid by a plastid targeting signal sequence. The isoprene synthases generally have a high Michaelis-Menten constant (Km), typically 1 millimolar or higher, and thus require high concentrations of dimethylallyl diphosphate to function efficiently.
Although microbes that naturally produce isoprene are known in the art (Kuzma, J., Nemecek-Marshall, M., Pollock, W. H., and R. Fall. 1995. Bacteria produce the volatile hydrocarbon isoprene. Curr. Microbiol. 30: 97-103; Wagner, W. P., Nemecek-Marshall, M., and R. Fall. 1999. Three distinct phases of isoprene formation during growth and sporulation of Bacillus subtilis. J. Bact. 181: 4700-4703; Fall, R. and S. D. Copley. 2000. Bacterial sources and sinks of isoprene, a reactive atmospheric hydrocarbon. Env. Microbiol. 2: 123-130; Xue, J., and B. K. Ahring. 2011. Enhancing isoprene production by the genetic modification of the 1-deoxy-D-xylulose-5-phosphate pathway in Bacillus subtilis. Appl. Env. Microbiol. 77: 2399-2405), the mechanism of isoprene production is unknown and the levels of isoprene production are relatively low. Several non-naturally occurring microorganisms have been engineered to produce isoprene, e.g., U.S. patent application Ser. No. 12/335,071, wherein isoprene production requires an isoprene synthase. For efficient function of isoprene synthase, high intracellular levels of dimethylallyl diphosphate are required; however, high levels of intracellular dimethylallyl diphosphate are also toxic to the cells, retarding growth and reducing the rates and yields of isoprene production (Martin, V. J. J., Pitera, D. J., Withers, S. T., Newman, J. D. and J. D. Keasling. 2003. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature Biotech. 21: 796-802; Withers, S. T., Gottlieb, S. S., Lieu, B., Newman, J. D., and J. D. Keasling. 2007. Identification of isopentenol biosynthetic genes from Bacillus subtilis by a screening method based on isoprenoid precursor toxicity. Appl. Env. Microbiol. 73: 6277-7283; Sivy, T. L., Fall, R., and T. N. Rosentiel. 2011. Evidence of isoprenoid precursor toxicity in Bacillus subtilis. Biosci. Biotechnol. Biochem. 75: 2376-2383). The problems associated with the direct chemical conversion of DMAPP to isoprene by isoprene synthases limits the potential for the biological production of commercially relevant amounts of isoprene.
Thus, there is a need for microorganisms and processes for the more efficient biological production of isoprene.